1. Field of the Invention
The present invention relates to the field of molecular biology. In particular, the present invention relates to the fields of storage, synthesis and amplification of nucleic acids. Specifically the invention relates to storage of RNA (particularly mRNA) on a solid matrix or support and to manipulation of the RNA by a number of molecular biology techniques including RT-PCR and cDNA synthesis (particularly cDNA library synthesis).
2. Related Art
The disclosures of the following applications were incorporated by reference into U.S. Provisional Application No. 60/175,307, filed Jan. 10, 2000, and are incorporated by reference into the present application: U.S. patent application Ser. No. 09/054,485, filed Apr. 3, 1998, now abandoned, which claims priority of U.S. provisional application 60/042,629, field Apr. 3, 1997, and the continuing application of Ser. No. 09/054,485, U.S. patent application Ser. No. 09/472,066, filed Dec. 23, 1999, now U.S. Pat. No. 6,495,350, issued Dec. 17, 2002; U.S. patent application Ser. No. 09/076,115, filed May 12, 1998, which claims priority of U.S. provisional application 60/046,219, filed May 12, 1997; U.S. patent application Ser. No. 09/354,664, filed Jul. 16, 1999, now U.S. Pat. No. 6,750,059, issued Jun. 15, 2004; and U.S. provisional application Ser. No. 60/122,395, filed Mar. 2, 1999.
Storage of Nucleic Acids
For many projects, generation of numerous DNA samples from biological specimens is routine. Handling and archiving a large collection can become a logistical problem for the laboratory. One solution, used in forensic labs, is the blood-storage medium FTA® Cards. The FTA® GeneCard is a chemically-treated filter paper designed for the collection and storage of biological samples for subsequent DNA analysis (1–3). It is suitable for storage of blood samples, as well as mammalian cells and tissues for PCR analysis and other genomic DNA applications (4). It is useful for recovery of plasmid DNA for PCR and transformation from archived bacterial cultures and colonies (5–6), as well as for storage and recovery of M13 phage for DNA sequencing applications (M. Goldsborough, personal communication).
An FTA® Card can be used to store genomic DNA in the form of dried spots of human whole blood, the cells of which were lysed on the paper. Stored at room temperature, genomic DNA on FTA® paper is reported to be stable at least 7.5 years (Burgoyne, et al., Conventional DNA Collection and Processing: Disposable Toothbrushes and FTA®Paper as a Non-threating Buccal-Cell Collection Kit Compatible with Automatable DNA Processing, 8th International Symposium on Human Identification, Sep. 17–20, 1997). Before analysis of the captured DNA, a few simple washing steps remove the stabilizing chemicals and cellular inhibitors of enzymatic reactions. Since the DNA remains with the paper, the manipulations to purify the DNA are simplified and amenable to automation. DNA samples on FTA® Cards offer a very compact archival system compared to glass vials or plastic tubes located in precious freezer space. Storage of RNA on dry solid medium is also described (see Burgoyne, U.S. Pat. No. 5,976,572).
Reverse Transcription of RNA
The term “reverse transcriptase” describes a class of polymerases characterized as RNA-dependent DNA polymerases. All known reverse transcriptases require a primer to synthesize a DNA transcript from an RNA template. Historically, reverse transcriptase has been used primarily to transcribe mRNA into cDNA which can then be cloned into a vector for further manipulation.
Avian myoblastosis virus (AMV) reverse transcriptase was the first widely used RNA-dependent DNA polymerase (Verma, Biochem. Biophys. Acta 473:1(1977)). The enzyme has 5′→3′ RNA-directed DNA polymerase activity, 5′→3′ DNA-directed DNA polymerase activity, and RNase H activity. RNase H is a processive 5′ and 3′ ribonuclease specific for the RNA strand for RNA-DNA hybrids (Perbal, A Practical Guide to Molecular Cloning, New York: Wiley & Sons (1984)). Errors in transcription cannot be corrected by reverse transcriptase because known viral reverse transcriptases lack the 3′→5′ exonuclease activity necessary for proofreading (Saunders and Saunders, Microbial Genetics Applied to Biotechnology, London: Croom Helm (1987)). A detailed study of the activity of AMV reverse transcriptase and its associated RNase H activity has been presented by Berger et al., Biochemistry 22:2365–2372 (1983).
Another reverse transcriptase which is used extensively in molecular biology is reverse transcriptase originating from Moloney murine leukemia virus (M-MLV). See, e.g., Gerard, G. R., DNA 5:271–279 (1986) and Kotewicz, M. L., et al., Gene 35:249–258 (1985). M-MLV reverse transcriptase substantially lacking in RNase H activity has also been described. See, e.g., U.S. Pat. No. 5,244,797.
PCR Amplification of RNA
Reverse transcriptases have been extensively used in reverse transcribing RNA prior to PCR amplification. This method, often referred to as RNA-PCR or RT-PCR, is widely used for detection and quantitation of RNA.
To attempt to address the technical problems often associated with RT-PCR, a number of protocols have been developed taking into account the three basic steps of the procedure: (a) the denaturation of RNA and the hybridization of reverse primer; (b) the synthesis of cDNA; and (c) PCR amplification. In the so-called “uncoupled” RT-PCR procedure (e.g., two-step RT-PCR), reverse transcription is performed as an independent step using the optimal buffer condition for reverse transcriptase activity. Following cDNA synthesis, the reaction is diluted to decrease MgCl2 and deoxyribonucleoside triphosphate (dNTP) concentrations to conditions optimal for Taq DNA Polymerase activity, and PCR is carried out according to standard conditions (see U.S. Pat. Nos. 4,683,195 and 4,683,202). In contrast, “coupled” RT-PCR methods use a common or compromised buffer for reverse transcriptase and Taq DNA Polymerase activities. In one version, the annealing of reverse primer is a separate step preceding the addition of enzymes, which are then added to the single reaction vessel. In another version, the reverse transcriptase activity is a component of the thermostable Tth DNA polymerase. Annealing and cDNA synthesis are performed in the presence of Mn++, then PCR is carried out in the presence of Mg++ after the removal of Mn++ by a chelating agent. Finally, the “continuous” method (e.g., one-step RT-PCR) integrates the three RT-PCR steps into a single continuous reaction that avoids the opening of the reaction tube for component or enzyme addition. Continuous RT-PCR has been described as a single enzyme system using the reverse transcriptase activity of thermostable Taq DNA Polymerase and Tth polymerase and as a two-enzyme system using AMVRT and Taq DNA Polymerase wherein the initial 65° C. RNA denaturation step was omitted.
cDNA and cDNA Libraries
In examining the structure and physiology of an organism, tissue or cell, it is often desirable to determine its genetic content. The genetic framework of an organism is encoded in the double-stranded sequence of nucleotide bases in the deoxyribonucleic acid (DNA) which is contained in the somatic and germ cells of the organism. The genetic content of a particular segment of DNA, or gene, is only manifested upon production of the protein which the gene encodes. In order to produce a protein, a complementary copy of one strand of the DNA double helix (the “coding” strand) is produced by polymerase enzymes, resulting in a specific sequence of ribonucleic acid (RNA). This particular type of RNA, since it contains the genetic message from the DNA for production of a protein, is called messenger RNA (mRNA).
Within a given cell, tissue or organism, there exist myriad mRNA species, each encoding a separate and specific protein. This fact provides a powerful tool to investigators interested in studying genetic expression in a tissue or cell—mRNA molecules may be isolated and further manipulated by various molecular biological techniques, thereby allowing the elucidation of the full functional genetic content of a cell, tissue or organism.
One common approach to the study of gene expression is the production of complementary DNA (cDNA) clones. In this technique, the mRNA molecules from an organism are isolated from an extract of the cells or tissues of the organism. This isolation often employs solid chromatography matrices, such as cellulose or Sepharose, to which oligomers of thymidine (T) have been complexed. Since the 3′ termini on all eukaryotic mRNA molecules contain a string of adenosine (A) bases, and since A binds to T, the mRNA molecules can be rapidly purified from other molecules and substances in the tissue or cell extract. From these purified mRNA molecules, cDNA copies may be made using an enzyme having reverse transcriptase (RT) activity, which results in the production of single-stranded cDNA molecules complementary to all or a portion of the mRNA templates. Incubating the single-stranded cDNA under appropriate conditions allows synthesis of double-stranded DNA which may then be inserted into a plasmid or a vector.
This entire process, from isolation of mRNA to insertion of the cDNA into a plasmid or vector to growth of host cell populations containing the isolated gene, is termed “cDNA cloning.” If cDNAs are prepared from a number of different mRNAs, the resulting set of cDNAs is called a “cDNA library,” an appropriate term since the set of cDNAs represents the different populations of functional genetic information (genes) present in the source cell, tissue or organism. Genotypic analysis of these cDNA libraries can yield much information on the structure and function of the organisms from which they were derived.
In traditional production methods, the cDNA molecules must be size fractionated and multiple phenol/chloroform extractions and ethanol precipitations performed. Each of these requirements has inherent disadvantages, such as product loss and limitations in cDNA yield due to multiple extractions/precipitations (Lambert, K. N., and Williamson, V. M., Nucl. Acids Res. 21(3):775–776 (1993)).
These disadvantages have been partially addressed in the literature. For example, several investigators have reported methods for the isolation of polyA+ mRNA from cell and tissue samples by binding the mRNA to latex or paramagnetic beads coupled with oligo(dT); single-stranded cDNA molecules may then be produced by reverse transcription of these immobilized mRNA molecules (Lambert, K. N., and Williamson, V. M., Nucl. Acids Res. 21(3):775–776 (1993); Kuribayashi-Ohta, K., et al., Biochim. Biophys. Acta 1156:204–212 (1993); Sasaki, Y. F., et al., Nucl. Acids Res. 22(6):987–992 (1994); Mészáros, M., and Morton, D. B., BioTechniques 20(3):413–419 (1996); Fellman, F., et al., BioTechniques 21(5):766–770 (1996)). Such solid phase synthesis methods are less prone to the yield limitations resulting from the extraction/precipitation steps of the traditional methods.
However, these methods still have several important limitations. For example, each of these methods relies on PCR amplification prior to cloning of the cDNA molecules, often resulting in biased cDNA libraries (i.e., highly expressed sequences predominate over those that are expressed in lower quantities). In addition, these methods often are less efficient than conventional cDNA synthesis methods which use solution hybridization of the primer-adapter to the template (i.e., rotational diffusion is required for increased hybridization rates; see Schmitz, K. S., and Schurr, J. M., J. Phys. Chem. 76:534–545 (1972); Ness, J. V., and Hahn, W. E., Nucl. Acids Res. 10(24):8061–8077 (1982)). Finally, the above-described techniques use heat or chemical denaturation to release the nascent cDNA molecules from the solid phase for further processing, which can result in product loss and/or damage.